Background. Molecular mechanisms underlying synaptic formation are one of the central themes of modern neuroscience. Best established role of acetylcholinesterase (AChE) is the termination of signal transmission in the neuromuscular junction (NMJ) and other cholinergic synapses by catalyzing hydrolysis of acetylcholine. Most of the AChE in the NMJ is bound to the specialized basal lamina located between the presynaptic membrane contributed by nerve ending and postsynaptic membrane belonging to the skeletal muscle fiber. Both cells can therefore be a source of synaptic AChE. Present evidencestrongly supports muscular origin of synaptic AChE, however the possibility of neural origin has never been eliminated and is supported by another line of evidence. Aim. The aim of this work was to study the origin ofsynaptic AChE at the earlier stages of the formation of mammalian NMJ under the in vitro conditions. Hzpothesis. At least part of the synaptic AChE, foundin the NMJ at the earlier stages of its formation is contributed by the motor neuron. Approach and methods. We employed an in vitro model in which human muscle was co-cultured with the explants isolated from the embryonic ratspinal cord. In these co-cultures the motor neurons originate from the rat spinal cord explants and form differentiated and long-lived NMJs with human muscle fibers. By using species specific antibodies against human and rat AChE, respectively, we were able to distinguish between AChE of muscle (human)and neural (rat) origin. Species specificity of antiAChE antibodies wastested by Western blotting. Phase-contrast microscopy and fluorescent identification of acetylcholine receptors were used for the visualization of the NMJs. (Abstract truncated at 2000 characters).